Human RAP74 is the large subunit of Transcription Factor IIF (TFIIF), an initiation and elongation factor for transcription by RNA polymerase II. This protein is highly phosphorylated in vivo. RAP74 has a somewhat unusual domain structure, with basic N- and C-terminal domains separated by a highly charged and overall acidic central region. The N-terminal domain is involved in binding to RAP30, the small subunit of TFIIF. The C-terminal domain binds to DNA and RNA polymerase II and affects the catalytic potential of polymerase in in vitro transcription assays. The central portion includes sites of phosphorylation and appears to be a regulatory hinge for the N- and C-terminal domains. Using casein kinase II in vitro, RAP74 is phosphorylated within the central portion of the molecule. These phosphorylation sites are being precisely located by peptide mass mapping using specific enzymatic digestions and MALDI-MS analysis. In vivo phosphorylated material will be similarly analyzed to determine whether the casein kinase sites are of physiological importance. Assays are being developed to analyze the importance of phosphorylation of RAP74 in transcription.